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L. S. Etheredge, B. Kane, N. Valdov, D. E. Birk, J. R. Hassell; Growth Factor Specific Extracellular Matrix Formation by Keratocyte Cultures Under Agarose. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5701.
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We previously found that IGF-I, TGF-β, and PDGF, but not FGF-2, stimulate collagen synthesis by keratocytes in culture. We also found that culturing insulin activated keratocytes under a thin layer of agarose increases the processing of procollagen to collagen and increases ECM formation (PMID: 18938157). We now evaluate the ECM formed by keratocytes cultured in these growth factors and under agarose.
Collagenase-isolated keratocytes from bovine corneas were plated at 40,000 cells/cm2 and then cultured with DMEM/F12 alone, or DMEM/F12 supplemented with either 10ng IGF-I, 2ng TGF-β, 10ng FGF-2, or 10ng PDGF/ml, all with ascorbate. Cultures were overlayed with ~1mm of 3% agarose on day 4 and harvested for analysis on day 12. Keratocytes cell number was determined by measuring DNA content (cyquant assay). Collagen was determined by pepsin digestion, SDS/PAGE, simply blue staining, and by western blots with antibodies to procollagen I and III. ECM morphology was evaluated by electron microscopy.
Compared to cultures without an agarose overlay, cultures with an agarose overlay had a 50%-89% reduction in levels of procollagen in the media. Conversely, agarose cultures had higher levels of collagen type I deposited with the cell layer, IGF-I was 9.1 fold higher; TGF-β was 6.2 fold higher; PDGF was 2.8 fold higher; DMEM/F12 was 2.5 fold higher; and FGF-2 was 0.6 fold higher. Electron microscopy showed that the FGF-2 agarose cultures had little ECM and the keratocytes were in close cell contact while IGF-I agarose cultures had the least cell contact with an extensive fibrillar ECM. TGF-β agarose cultures had both a fibrillar and amorphous ECM.
The results of this study show that the agarose overlay increases ECM formation with the cell layer only when the synthesis of collagen was stimulated and that the ECM morphology is growth factor specific.
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